Page 1 of 38 Diabetes 1 Metabolic rescue of obese adipose-derived stems cells by Lin28/Let7 pathway. Laura M. Pérez1, Aurora Bernal1, Nuria San Martín1, Margarita Lorenzo2,†, Sonia Fernández-Veledo3,4 and Beatriz G. Gálvez1. 1 Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain. 2 Facultad de Farmacia, Universidad Complutense de Madrid, Spain. 3 University Hospital of Tarragona Joan XXIII. Pere Virgili Institute and Rovira i Virgili University, Tarragona (Spain). 4CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid (Spain). Running title: Adipose stem cell metabolism Word count: 5,547. Figures number: 7 + 4 Suppl. Corresponding author: Beatriz G. Gálvez Centro Nacional de Investigaciones Cardiovasculares C/Melchor Fernández Almagro, 3. 28029-Madrid. Spain. Phone: +34 914531200 Fax: +34 914531240 E-mail: [email protected] Diabetes Publish Ahead of Print, published online February 19, 2013 Diabetes Page 2 of 38 2 ABSTRACT Adipose-derived stem cells (ASCs) are promising candidates for autologous cell-based regeneration therapies by virtue of their multilineage differentiation potential and immunogenicity, however relatively little is known about their role in adipose tissue physiology and dysfunction. Here we evaluated whether ASCs isolated from non-obese and obese tissue differed in their metabolic characteristics and differentiation potential. During differentiation to mature adipocytes, both mouse and human ASCs derived from non-obese tissues increased their insulin sensitivity and inhibition of lipolysis while obese-derived ASCs were insulin-resistant, showing impaired insulin-stimulated glucose uptake and resistance to the antilipolytic effect of insulin. Furthermore, obese-derived ASCs showed enhanced release of proinflammatory cytokines and impaired production of adiponectin. Interestingly, the delivery of cytosol from control ASCs into obese-derived ASCs using a lipid- based protein-capture methodology restored insulin sensitivity on glucose and lipid metabolism and reversed the proinflammatory cytokine profile, in part due to the restoration of Lin28 protein levels. In conclusion, glucose and lipid metabolism as well as maturation of ASCs are truncated in an obese environment. The reversal of the altered pathways in obese cells by delivery of normal subcellular fractions offers a potential new tool for cell therapy. Keywords: obesity, adipocyte, mesenchymal precursors, reprogramming, metabolism. INTRODUCTION Adipose tissue is now recognized as an important endocrine and metabolic organ that, when accumulated in excess, increases the risk of chronic diseases such as diabetes, stroke and arterial hypertension (1). Recently, new mechanisms that control the obesity phenotype have been identified, such as the equilibrium between white and brown adipose tissue, the localization of adipose mass (visceral versus ventral), and the presence of adipose and mesenchymal stem cells (1-4). Although the relative importance of fat tissue type and localization are being actively unraveled, the role of stem cells in adipose tissue physiology and dysfunction is still poorly understood. Adult stem cells are multipotent cells that contribute to the homeostasis of various organs, including Page 3 of 38 Diabetes 3 adipose tissue. Adipose stem cells (ASCs) are a class of mesenchymal stem cells (MSCs) localized in adipose tissue that have attracted increasing interest because of their potential to differentiate into adipogenic, osteogenic, chondrogenic and other mesenchymal lineages (5-8). Other clinically attractive properties attributed to ASCs include pro-angiogenic and anti-inflammatory actions (9-11). Moreover, depending on the environmental conditions, ASCs can be beneficial or detrimental to health. ASCs thus represent possible target for therapies aimed at modulating the response of the body to obesity and diabetes, as well as a potential tool for regenerative medicine. Adipocytes are central to the control of energy balance and lipid homeostasis (12). In response to prolonged obesity, adipocytes become hypertrophic, and new adipocytes are required to counter the metabolic dysfunction of the hypertrophic cells (13; 14). It has been postulated that the adipose tissue depots of obese individuals have already committed all of their stem cell reserves to the adipocyte lineage, and therefore have no capacity to generate new adipocytes (15-17). In this study, we demonstrate that the differentiation of mouse and human adipose mesenchymal stem cells into mature well-functioning adipocytes is truncated in an obese environment, resulting in impaired metabolic function. We also validate a novel approach to restore normal adipocyte metabolic responsiveness in obese-derived stem cells by cytosolic transfer. RESEARCH DESIGN AND METHODS Reagents Culture media and anti-Let-7 microRNA inhibitor were purchased from Invitrogen (Paisley, UK). 2-deoxy- D[1-3H]glucose (11.0 Ci/mmol) was from GE Healthcare (Rainham, U.K.). Antibodies to Glut-4, IRS1 and phospho-IRS1 (Tyr612) and anti-caveolin-1, anti-β-tubulin and anti-Pi3-Kinase were purchased from Millipore (Bedford, MA). An antibody to TNF-α and recombinant TNF-α cytokine were purchased from BD Pharmingen (Franklin Lakes, NJ). An antibody to MCP-1 was purchased from R&D Systems (Minneapolis, MN) and recombinant MCP-1 cytokine was from Reprokine (Valley Cottage, NJ). An antibody to Lin28b was purchased from Abcam (Cambridge, UK), while purified full length Lin28b protein was obtained from Applied Biological Materials Inc. (Richmond, Canada). The mirCury LNA microRNA Let-7 inhibitor was purchased from Exiqon Diabetes Page 4 of 38 4 (Vedbaek, Denmark). Unless otherwise stated, all other reagents were purchased from Sigma Aldrich (Poole, Dorset, UK). Animals C57BL/6 mice and leptin deficient Ob/Ob mice (18) were obtained from Charles River (Wilmington, MA) and maintained and used in accordance with the National Institutes of Health Animal Care and Use Committee. Mice were sacrificed by cervical dislocation for sample collection. DIO rodent purified high fat diet (formula 58Y1) was obtained from TestDiet (IPS Product Supplies Ltd, London, UK). Isolation of mouse and human adipocyte stem cells Adipocyte stem cells (ASCs) were isolated by the explant method (19). Briefly, small pieces of subcutaneous adipose tissue were collected from five control and five obese mice (four months old) and placed on gelatin- coated plates (Fig 1A). After 7 days rounded cells emerging from the explants were selected, cloned by limiting dilution and grown to obtain ASCs. Independent ASC clonal lines obtained from five different mice were first characterized by morphology (Fig 1A) and by flow cytometry for the surface antigens that have been used to specifically define this population (3), namely Sca-1+, CD34+, CD44+, CD29+ and CD45- (Fig 1B). These clones were named cASCs or oASCs for cells derived from control or obese mice, respectively. We obtained two clones from each of the five obese mice, obtaining a total of ten oASCs clones; with control explants, we obtained ten independent clones from each five animals. Finally, five oASC and five cASC clones derived from independent animals were selected randomly and as all expressed similar surface markers and presented similar morphology, pooled respectively as a population of oASC or cASC and used for experiments. ASCs were maintained on gelatin-coated plates in DMEM containing 10% FBS and supplemented with glutamine and pen/step. Human adipose samples were obtained from patients after bariatric surgery. Informed consent was obtained from all subjects and the sample collection conformed to the principles set out in the WMA Declaration of Helsinki and the NIH Belmont Report. Adipose stem cells were isolated from a total of five control (BMI <22) and five obese (BMI>30) patients by the explant method, similar to the protocol used above for mice (19; 20). Page 5 of 38 Diabetes 5 Human ASCs were also characterized by surface marker expression and named as hASCs. Human primary adipose stem cells were also purchased from Lonza (Walkersville, MD); one derived from an obese patient with a BMI=31 (PT5006 lot 4308 stock 32) and the other from a non-obese patient (BMI<22) (PT4504 lot 4028 stock 27). All cells were maintained in the medium supplied with the PT4505-ADSC Bullekit (Lonza). Cell culture and treatments To induce adipogenic differentiation, ASCs were cultured in serum-free DMEM/F12 medium (1:1) supplemented with 10 µg/ml transferrin, 15 mM NaHCO3, 15mM HEPES, 33 µM biotin, 17 µM pantothenate, 1 nM insulin, 20 pM triiodothyronine, 1 µM cortisol, plus antibiotics. Accumulation of triglycerides in adipocytes was visualized by staining formalin-fixed cells with Oil Red O. Triglyceride accumulation was assessed microscopically, and Oil Red O concentration was quantified spectrophotometrically at 510 nm. Lipid content was also analyzed enzymatically with a triglyceride determination kit. In order to measure the osteogenic potential of the ASCs, 2 x 104 cells were incubated in DMEM containing 10% FBS and supplemented with glutamine and pen/step until a confluent layer was achieved and then osteogenic medium was added, containing IMDM supplemented with 9 % FBS, 9 % HS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 ng/mL L-thyroxine, 20 mM β-glycerol phosphate, 100 nM dexamethansone, and 50 µM ascorbic acid. Medium was refreshed every 3-4 d. After 17 d of culture cells were fixed in 10 % formalin